Tumor stiffness has been implicated as a driving force in tumor progression and metastasis. Matrix stiffness has been shown to alter the phenotype of breast epithelial cells from growth-arrested in soft conditions to malignant and invasive in stiff conditions. However, it is unknown whether invading cells retain `memory' of stiff environments after dissemination from the primary tumor, and if so, by what mechanism. This proposal aims to determine the extent and basis of mechanical memory of stiff microenvironments in mammary epithelial cells. Using a novel 3D model in which matrix stiffness can be tuned independently of ligand density, polarized, growth arrested mammary acini can be generated in soft gels while stiff gels give rise to malignant, invasive phenotypes. After culture in either soft or stiff conditions, the gel stiffness will be altered to determine the reversibility of the phenotypes initially established. These experiments will demonstrate whether mechanical memory exists for breast epithelial cells or in cancer contexts in general. Next chromatin accessibility and transcription factor occupancy will be assessed genome-wide using a transposition-based assay (ATAC-seq) suited for low cells numbers required for 3D culture. Epigenetic modifiers will be screened based on candidates identified from epigenomic data to identify the molecular mechanism driving stiffness-induced chromatin remodeling. Successful completion of this proposal will reveal, for the first time, the extent of mechanical memory in a cancer context, and the epigenomic landscape of cells in 3D culture.